Interferons (IFNs) exhibit important antineoplastic properties in vitro and in vivo and are key elements in the immune surveillance against cancer. These cytokines have widespread applications in clinical medicine, but their utility in the treatment of many malignancies is frequently limited by the development of neoplastic cell resistance. We have identified a novel IFN-signaling cascade, involving the kinases Mnk1 and Mnk2. Our data have established that Mnk kinases play key roles in IFN-dependent mRNA translation and growth suppression, providing a direct link between MAPK pathways and mRNA translation of IFN-stimulated genes (ISGs). The engagement of this pathway ultimately regulates expression of protein products with key functional roles in the IFN-system, such as ISG15 which mediates ISGylation and other proteins with growth inhibitory properties. The current proposal is a systematic approach to establish the roles of Mnk pathways in the generation of IFN-mediated responses and to define the importance of defects in the activation of this cascade in the development of IFN-resistance in malignant cells. Specific aim 1 will identify upstream effector mechanisms and signaling events involved in the regulation of the Mnk pathway. Studies to define the roles of IFNR-associated Jak kinases in the activation of Mnk 1/2 will be performed, while the potential regulatory effects of Jak2 on the function of nuclear Mnk2b and mRNA nuclear export will be determined. The contribution of distinct MAPKKKs and MAPKKs in engagement of Mnks by the IFNR will be also assessed. Specific aim 2 will identify downstream elements of the pathway and will dissect their roles in the generation of IFN-dependent antineoplastic effects. The functional relevance of eIF4E phosphorylation and the involvement of hnRNPA1 and Sprouty proteins (Spry1 and 2) in IFN-signaling will be examined. Studies will be also performed to determine whether Mnks regulate mRNA translation of SLFN genes, a novel family of cell-cycle regulators that we have recently identified as mediators of IFN-antiproliferative responses. Specific aim 3 will determine the role of Mnk kinases in the generation of the suppressive effects of IFNa in Ph (-) myeloproliferative neoplasms with the JAK2V617F mutation. The requirement of Mnk activity in the induction of IFNa effects on primary clonal hematopoietic progenitors from patients with P. Vera and ET will be examined;and the ability of IFNa to induce responses in JAK2V617F mouse models with targeted disruption of the Mnk1 and/or Mnk2 genes will be determined. Altogether, these studies will advance our understanding of the mechanisms of generation of IFN-dependent antileukemic responses;provide important information on the events that lead to malignant cell resistance;and form the basis for the development of novel antineoplastic agents and approaches to overcome such resistance. PUBLIC HEALTH RELEVANCE: Interferons exhibit important antitumor effects in vitro and in vivo. These cytokines play key roles in the immune surveillance against cancer and have important activities in certain clinical settings, but the precise mechanisms by which they generate their effects remain to be defined. We have identified a novel IFN- activated signaling cascade involving Mnk kinases, which plays an important role in mRNA translation of IFN stimulated genes (ISGs), including genes with important biological properties, such Isg15 and Isg54. We have also identified a novel group of ISGs (SLFN genes) that regulate cell cycle progression and mediate IFN- dependent growth inhibitory responses. Our data suggest a model by which IFN-activated Mnk kinases complement the function of Jak-Stat pathways by regulating signals for mRNA translation of Stat-activated genes. The current proposal will precisely define the role of the Mnk pathway in the generation of the antileukemic properties of IFNs and will identify downstream effectors and targets of this cascade. Studies will be also performed to examine whether defects in the activation of Mnk kinases and/or their effectors result in leukemic cell resistance to the effects of IFNs in vitro and in vivo.